Increasing the oil content in plants and, in particular, in plant seeds is of great interest for traditional and modern plant breeding and in particular for plant biotechnology. Owing to the increasing consumption of vegetable oils for nutrition or industrial applications, possibilities of increasing or modifying vegetable oils are increasingly the subject of current research (for example Töpfer et al. (1995) Science 268:681-686). Its aim is in particular increasing the fatty acid content in seed oils.
The fatty acids which can be obtained from the vegetable oils are also of particular interest. They are employed, for example, as bases for plasticizers, lubricants, surfactants, cosmetics and the like and are employed as valuable bases in the food and feed industries. Thus, for example, it is of particular interest to provide rapeseed oils with fatty acids with medium chain length since these are in demand in particular in the production of surfactants.
The targeted modulation of plant metabolic pathways by recombinant methods allows the modification of the plant metabolism in an advantageous manner which, when using traditional breeding methods, could only be achieved after a complicated procedure or not at all. Thus, unusual fatty acids, for example specific polyunsaturated fatty acids, are only synthesized in certain plants or not at all in plants and can therefore only be produced by expressing the relevant enzyme in transgenic plants (for example Millar et al. (2000) Trends Plant Sci 5:95-101).
Triacylgylcerides and other lipids are synthesized from fatty acids. Fatty acid biosynthesis and triacylglyceride biosynthesis can be considered as separate biosynthetic pathways owing to the compartmentalization, but as a single biosynthetic pathway in view of the end product. Lipid synthesis can be divided into two part-mechanisms, one which might be termed “prokaryotic” and another which may be termed “eukaryotic” (Browse et al. (1986) Biochemical J 235:25-31; Ohlrogge & Browse (1995) Plant Cell 7:957-970). The prokaryotic mechanism is localized in the plastids and encompasses the biosynthesis of the free fatty acids which are exported into the cytosol, where they enter the eukaryotic mechanism in the form of fatty acid acyl-CoA esters and are esterified with glycerol-3-phosphate (G3P) to give phosphatidic acid (PA). PA is the starting point for the synthesis of neutral and polar lipids. The neutral lipids are synthesized on the endoplasmic reticulum via the Kennedy pathway (voelker (1996) Genetic Engineering, Setlow (ed.) 18:111-113; Shankline & Cahoon (1998) Annu Rev Plant Physiol Plant Mol Biol 49:611-649; Frentzen (1998) Lipids 100:161-166). Besides the biosynthesis of triacylglycerides, G3P also plays a role in glycerol synthesis (for example for the purposes of osmoregulation and against low-temperature stress for example).
GP3, which is essential for the synthesis, is synthesized here by the reduction of dihydroxyacetone phosphate (DHAP) by means of glycerol-3-phosphate dehydrogenase (G3PDH), also termed dihydroxyacetone phosphate reductase. As a rule, NADH acts as reducing cosubstrate (EC 1.1.1.8). A further class of glycerol-3-phosphate dehydrogenases (EC 1.1.99.5) utilizes. FAD as cosubstrate. The enzymes of this class catalyze the reaction of DHAP to G3P. In eukaryotic cells, the two classes of enzymes are distributed in different compartments, those which are NAD-dependent being localized in the cytosol and those which are FAD-dependent being localized in the mitochondria (for Saccharomyces cerevisiae, see, for example, Larsson et al., 1998,. Yeast 14:347-357). EP-A 0 353 049 describes an NAD-independent G3PDH from Bacillus sp. In Saccharomyces cerevisiae too, an NAD-independent G3PDH is identified (Miyata K, Nagahisa M (1969) Plant Cell Physiol 10(3):635-643).
G3PDH is an essential enzyme in prokaryotes and eukaryotes which, besides having a function in lipid biosynthesis, is one of the enzymes responsible for maintaining the cellular redox status by acting on the NAD+/NADH ratio. Deletion of the GPD2 gene in Saccharomyces cerevisiae (one of two G3PDH isoforms in this yeast) results in reduced growth under anaerobic conditions. In addition, G3PDH appears to play a role in the stress response of yeast mainly to osmotic stress. Deletion of the GPD1 gene in Saccharomyces cerevisiae causes hypersensitivity to sodium chloride.
Sequences for G3PDHs have been described for insects (Drosophila melanogaster, Drosophila virilis), plants (Arabidopsis thaliana, Cuphea lanceolata), mammals (Homo sapiens, Mus musculus, Sus scrofa, Rattus norvegicus), fish (Salmo salar, Osmerus mordax), birds (Ovis aries), amphibians (Xenopus laevis), nematodes (Caenorhabditis elegans), algae and bacteria.
Plant cells have at least two G3PDH isoforms, a cytoplasmic isoform and a plastid isoform (Gee R W et al. (1988) Plant Physiol 86:98-103; Gee R W et al. (1988) Plant Physiol 87:379-383). In plants, the enxymatic activity of glycerol-3-phosphate dehydrogenase was first found in potato tubors (Santora G T et al. (1979) Arch Biochem Biophys 196:403-411). Further G3PDH activities which were localized in the cytosol and the plastids were detected in other plants such as peas, maize or soya (Gee R W et al. (1988) PLANT PHYSIOL 86(1): 98-103). G3PDHs from algae such as, for example, two plastid G3PDH isoforms and one cytosolic G3PDH isoform from Dunaliella tertiolecta have furthermore been described (Gee R et al.(1993) Plant Physiol 103(1):243-249; Gee R et al. (1989) PLANT PHYSIOL 91(1):345-351). As regards the plant G3PDH from Cuphea lanceolata, it has been proposed to obtain an increased oil content or a shift in the fatty acid pattern-by overexpression in plants (WO 95/06733). However, such effects have not been proven.
Bacterial G3PDHs and their function have been described (Hsu and Fox (1970) J Bacteriol 103:410-416; Bell (1974) J Bacterial 117:1065-1076).
WO 01/21820 describes the heterologous expression of a mutated E. coli G3PDH for increased stress tolerance and modification of the fatty acid composition in storage oils. The mutated E. coli G3PDH (gpsA2FR) exhibits a single amino acid substitution which brings about reduced inhibition via G3P. The heterologous expression of the gpsA2FR mutant leads to glycerolipids with an increased C16 fatty acid content and, accordingly, a reduced C18 fatty acid content. The modifications in the fatty acid pattern are relatively minor: an increase of 2 to 5% in the 16:0 fatty acids and of 1.5 to 3.5% in the 16:3 fatty acids, and a reduction in 18:2 and 18:3 fatty acids by 2 to 5% were observed. The total glycerolipid content remained unaffected.
G3PDHs from yeasts (Ascomycetes) such as    a) Schizosaccharomyces pombe (Pidoux AL et al. (1990) Nucleic Acids Res 18 (23): 7145; GenBank Acc.-No.: X56162; Ohmiya R et al. (1995) Mol Microbiol 18(5):963-73; GenBank Acc.-No.: D50796, D50797),    b) Yarrowia lipolytica (GenBank Acc.-No.: AJ250328)    c) Zygosaccharomyces rouxii (Iwaki T et al. Yeast (2001) 18(8):737-44; GenBank Acc.-No: AB047394, AB047395, AB047397) or    d) Saccharomyces cerevisiae (Albertyn J et al. (1994) Mol Cell Biol 14(6):4135-44; Albertyn J et al. (1992) FEBS LETT 308(2):130-132; Merkel J R et al. (1982) Anal Biochem 122 (1):180-185; Wang H T et al. (1994) J Bacteriol. 176(22):7091-5; Eriksson P et al. (1995) Mol Microbiol. 17(1):95-107; GenBank Acc.-No.: U04621, X76859, Z35169).    e) Emericella nidulans (GenBank Acc.-No.: AF228340)    f) Debaryomyces hansenii (GenBank Acc.-No.: AF210060) are furthermore described.